Methods to Assess Translocation

In a number of experimental situations it is desirable to translocate proteins from the exterior into the cytosol, and this may also be desirable for vaccination purposes. Since many proteins retain their function in fusion proteins, cDNA for the protein in question may be linked to the cDNA of a toxin A-moiety and, after expression and reconstitu- 

In experiments on transmembrane translocation of fusion proteins it is necessary to have a reliable and convenient way of monitoring translocation of the fusion protein. Measuring the toxic effect of the fusion protein provides an indication of translocation, but is not sufficient, since it only shows that the enzymatic A-fragment has been translocated. Thus, the passenger peptide or protein could have been removed proteolytically before translocation takes place. It should also be noted that monitoring toxicity is not very accurate in assessing how much A-fragment (or fusion protein) is translocated. 

A number of more direct methods can be used to demonstrate that the fusion protein has indeed been translocated  

The fusion protein can be supplied with a signal recognized by enzymes or other molecules exclusively present in the cytosol. 

The modified protein usually migrates slightly more rapidly in SDS-PAGE than the unmodified molecule (Fig. 4). 

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